Following tissue injury, exposition of membrane-bound Tissue Factor (TF), is a crucial step in the initiation of blood coagulation. TF binds to blood coagulation Factor VIIa, and the binary Factor VIIa/TF complex then activates Factor X to Factor Xa, leading to thrombin generation and fibrin formation (Broze, G. J. Jr. et al. 1988 Blood 71:335–343; Pedersen, A. H. et al. 1990 J Biol Chem 254:16786–16793; Rao, L. V. M., and Rapaport, S. I. 1987 Blood 69:645–651). Initiation of blood coagulation by Factor VIIa/TF is under control of tissue factor pathway inhibitor (TFPI) (Pedersen, A. H. et al. 1990 J Biol Chem 254:16786–16793; Rao, L. V. M., and Rapaport, S. I. 1987 Blood 69:645–651; Broze, G. J. Jr, and Miletich, J. P. 1987 PNAS USA 84:1886–1890; Rapaport, S. I. 1989 Blood 73:359–365), a 34- to 43-kDa multidomain protein with an acidic aminoterminus, three typical Kunitz-type inhibitor domains, and a basic carboxy terminus (Girard, T. J. et al. 1990 Science 248:1421–1424: Wun, T-C. et al. 1988 J Biol Chem 263:6001–6004). The second Kunitz domain binds and inhibits Factor Xa, and the first Kunitz domain binds to Factor VIIa/TF through formation of a final quarternary inhibitory complex consisting of Factor Xa-TFPI-Factor VIIa/TF (Lindhout, T., Fransen, J., and Willems, G. 1995 Thromb Haemost 74:910–915; Hamamoto, T. et al 1993 J Biol Chem 268:8704–8710; Broze, G. J. et al. 1990 Biochemistry 29:7539–7546; Rao, L. V. M., and Ruf, W. 1995 Biochemistry 34:10867–10871;. Baugh, R. J. et al. 1998 J Biol Chem 273:4378–4386; Wesselschmidt, R. et al. 1992 Blood 79:2004–2010). In addition to TFPI and other physiologic inhibitors of blood coagulation (e.g., antithrombin), a number of coagulation inhibitors from the salivary gland of blood-sucking invertebrates have been characterized (Law, L. H., Ribeiro, J. M., and Wells, M. A. 1992 Annu Rev Biochem 61:87–111; Markwardt, F. 1994 Pharmazie 49:313–316).
Ticks, such as Ixodes scapularis, are ectoparasites that feed for several days with their mouthparts embedded in their vertebrate hosts. Ixodes scapularis is a vector of Lyme disease. Lyme disease is a spirochetal illness caused by Borrelia burgdorferi, which is injected into the vertebrate host together with tick saliva. A number of pharmacologic properties have been described in I. scapularis saliva including inhibitors of neutrophil function (Ribeiro, J. M. C., Weis, J. J., and Telford, S. R. III 1990 Exp Parasitol 70:382–388), anticomplement (Ribeiro, J. M. C., 1987 Exp Parasitol 64:347–353; Valenzuela, J. G. et al. 2000 J Biol Chem 275:18717–18723), and anaphilatoxin-inactivating activities (Ribeiro, J. M. C., and Spielman A. 1986 Exp Parasitol 62:292–297) in addition to antiinflammatory and immunosuppresive components (Ribeiro, J. M. C. et al. 1985 J Exp Med 161:332–344). Antihemostatic compounds have also been molecularly characterized in the soft tick O. moubatta, including a platelet integrin αIIbβ3 integrin antagonist (Karczewski, J. et al. 1994 J Biol Chem 269:6702–6708) and inhibitors of blood coagulation such as ornithodorin, a thrombin inhibitor (Locht, A. et al. 1996 EMBO J 15:6011–6017), and tick anticoagulant peptide, a Factor Xa inhibitor (Waxman, L. et al. 1990 Science 248:593–596). The presence of thrombin and Factor Xa inhibitors seems to be a successful strategy evolutionarily developed by ticks to successfully feed on blood, because both molecules play an essential role in two key steps necessary for maintenance of the blood coagulation cascade (Jenny, N. S., and Mann K. G. in: Thrombosis and Hemorrhage, 2d ed. 1998, Loscalzo J, Schafer AI eds. Baltimore, Md.: Williams and Wilkins 1998 p 3; Davie, E. W. et al. 1991 Biochemistry 30:10363–10370) however, an inhibitor of Factor VIIa/TF has not yet been molecularly characterized.